Various publications, which may include patents, published applications, technical articles and scholarly articles, are cited throughout the specification in parentheses, and full citations of each may be found at the end of the specification. Each of these cited publications is incorporated by reference herein, in its entirety.
Nuclear receptors are members of a superfamily of transcription factors. The members of this family share structural similarities and regulate a diverse set of biological effects (Olefsky 2001). Ligands activate or repress these transcription factors that control genes involved in metabolism, differentiation and reproduction (Laudet and Gronmeyer 2002). Presently, the human genome project has identified about 48 members for this family and cognate ligands have been identified for about 28 of them (Giguere 1999). This protein family is composed of modular structural domains that can be interchanged within the members of the family without loss of function. A typical nuclear receptor contains a hypervariable N-terminus, a conserved DNA binding domain (DBD), a hinge region, and a conserved ligand binding domain (LBD). The function of the DBD is targeting of the receptor to specific DNA sequences (nuclear hormone response elements or NREs). The function of the LBD is recognition of its cognate ligand. Within the sequence of the nuclear receptor there are regions involved in transcriptional activation. The AF-1 domain is situated at the N-terminus and constitutively activates transcription (Rochette-Egly, Gaub et al. 1992; Rochette-Egly, Adam et al. 1997), while the AF-2 domain is embedded within the LBD and its transcriptional activation is ligand dependent (Wurtz, Bourguet et al. 1996). Nuclear receptors can exist as monomers, homodimers or heterodimers and bind to direct or inverted nucleotide repeats (Aranda and Pascual 2001; Laudet and Gronmeyer 2002).
The members of this family exist either in an activated or repressed basal biological state. The basic mechanism of gene activation involves ligand dependent exchange of co-regulatory proteins. These co-regulatory proteins are referred to as co-activators or co-repressors (McKenna, Lanz et al. 1999). A nuclear receptor in the repressed state is bound to its DNA response element and is associated with co-repressor proteins that recruit histone de-acetylases (HDACs) (Jones and Shi 2003). In the presence of an agonist there is an exchange of co-repressors with co-activators that in turn recruit transcription factors that assemble into an ATP dependent chromatin-remodeling complex. Histones are hyper-acetylated, causing the nucleosome to unfold, and repression is alleviated. The AF-2 domain acts as the ligand dependent molecular switch for the exchange of co-regulatory proteins. In the presence of an agonist the AF-2 domain undergoes a conformational transition and presents a surface on the LBD for interaction with co-activator proteins. In the absence of an agonist or in the presence of an antagonist the AF-2 domain presents a surface that promotes interactions with co-repressor proteins. The interaction surfaces on the LBD for both co-activators, and co-repressors overlap and provide a conserved molecular mechanism for gene activation or repression that is shared by the members of this family of transcription factors (Xu, Stanley et al. 2002).
Natural ligands that modulate the biological activity of nuclear receptors have been identified for only approximately one half of known nuclear receptors. Receptors for which no natural ligand has been identified are termed “orphan receptors”. The discovery of ligands or compounds that interact with an orphan receptor will accelerate the understanding of the role of the nuclear receptors in physiology and disease and facilitate the pursuit of new therapeutic approaches. A sub-class of these receptors, for which no natural ligands have been identified, is the estrogen related receptors (ERRs).
Estrogen Related Receptor alpha (ERR-α), also known as ERR-1, is an orphan receptor and was the first to be identified of the three members of the estrogen receptor related subfamily of orphan nuclear receptors (ERR-α, β, γ). The ERR subfamily is closely related to the estrogen receptors (ER-α and ER-β). ERR-α and ERR-β were first isolated by a low stringency hybridization screen (Giguere, Yang et al. 1988) followed later with the discovery of ERR-γ (Hong, Yang et al. 1999). The ERRs and ERs share sequence similarity with the highest homology observed in their DBDs, approximately 60%, and all interact with the classical DNA estrogen response element. Recent biochemical evidence suggested that the ERRs and ERs share co-regulator proteins and also target genes, including pS2, lactoferin, aromatase, and osteopontin (Hong, Yang et al. 1999; Zhang and Teng 2000; Giguere 2002; Kraus, Ariazi et al. 2002). It has been suggested that one of the main functions of ERRs is to regulate the response of estrogen responsive genes. The effects of the steroid hormone estrogen are primarily mediated in the breast, bone and endometrium, so it is reasonable to believe that compounds that interact with ERRs may find use for the treatment of bone related disease, breast cancer, and other diseases related to the reproduction system.
For example, it has been shown that ERR-α is present in both normal and cancerous breast tissue (Ariazi, Clark et al. 2002). It has also been reported that the main function of ERR-α in normal breast tissue is that of a repressor for estrogen responsive genes. In breast cancers or cell lines that are non-estrogen responsive (ER-α negative), ERR-α has been reported to be in an activated state (Ariazi, Clark et al. 2002). Therefore compounds that interact with ERR-α may be useful agents for the treatment of breast cancer that is ER-α negative and non-responsive to classical anti-estrogenic therapy, or may be used as an adjunct agent for anti-estrogen responsive breast cancers. These agents may act as antagonists by reducing the biological activity of ERR-α in these particular tissues.
Regarding bone related diseases, many post-menopausal women experience osteoporosis, a condition that has been clearly associated with a reduction of estrogen production. For example, it has been shown that reduction of estrogen levels results in increased bone loss (Turner, Riggs et al. 1994). It has also been shown that administration of estrogens to postmenopausal patients with osteoporosis has an anabolic effect on bone development (Pacifici 1996). The molecular mechanism linking estrogen receptors to bone loss is not well understood, however, since ER-α and ER-β knock-out animals have only minor skeletal defects (Korach 1994; Windahl, Vidal et al. 1999). With regard to ERR-α in bone, ERR-α expression has been shown to be regulated by estrogen (Bonnelye, Vanacker et al. 1997; Bonnelye, Merdad et al. 2001) and ERR-α expression is known to be maintained throughout stages of osteoblast differentiation. Furthermore, over-expression of ERR-α in rat calvaria osteoblasts, an accepted model of bone differentiation, resulted in an increase of bone nodule formation and treatment of rat calvaria osteoblasts with ERR-α antisense results in a decrease of bone nodule formation. ERR-α also regulates osteopontin, a protein believed to be involved in bone matrix formation. Therefore, compounds that modulate ERR-α by increasing its activity may have an anabolic effect for the regeneration of bone density and provide a benefit over current approaches that prevent bone loss. Such compounds may enhance the activity of the receptor by enhancing the association of the receptor with proteins that increase its activity or improve the stability of the receptor or by increasing the intracellular concentrations of the receptor and consequently increasing its activity. Conversely, with respect to bone diseases that are a result of abnormal bone growth, compounds that interact with ERR-α and decrease its biological activity may provide a benefit for the treatment of these diseases by retarding bone growth. Antagonism of the association of the receptor with co-activator proteins decreases the activity of the receptor.
ERR-α is also present in cardiac, adipose, and muscle tissue and forms a transcriptionally active complex with the PGC-1 co-activator family, which are co-activators implicated in energy homeostasis, mitochondria biogenesis, hepatic gluconeogenesis and in the regulation of genes involved in fatty acid beta-oxidation (Kamei, Ohizumi et al. 2003). ERR-α regulates the expression of medium chain acyl-CoA dehydrogenase (MCAD) through interactions with its promoter. MCAD is a gene involved in the initial reaction in fatty acid beta-oxidation. It is believed that in the adipose tissue, ERR-α regulates energy expenditure through the regulation of MCAD (Sladek, Bader et al. 1997; Vega and Kelly 1997). In antisense experiments in rat calvaria osteoblasts, in addition to the inhibition of bone nodule formation, there was an increase in adipocyte differentiation markers including aP2 and PPAR-γ (Bonnelye, Kung et al. 2002). An ERR-α knockout model has been described that exhibited reduced fat mass relative to the wild type. DNA chip analysis indicated that the ERR-α knockout mice have an alteration in the expression levels of genes involved in adipogenesis and energy metabolism (Luo, Sladek et al. 2003). More recently it has been shown that ERR-α regulates the expression of endothelial nitric oxide synthase, a gene that has a protective mechanism against arteriosclerosis (Sumi and Ignarro 2003). The biochemical evidence supports the involvement of ERR-α in metabolic homeostasis and differentiation of cells into adipocytes. Therefore, compounds interacting with ERR-α may affect energy homeostasis and provide a benefit for the treatment of obesity and metabolic syndrome related disease indications, including arteriosclerosis and diabetes (Grundy, Brewer et al. 2004).
Lion Bioscience AG disclosed the use of certain pyrazole derivatives as antagonists of ERR-α for treating cancer, osteoporosis, obesity, lipid disorders and cardiovascular disorders and for regulating fertility (US20060148876). Still other small molecules were also disclosed as ERR-α modulators (US20060014812; US20080221179).
There is a continuing need for new ERR-α inverse agonists that may find use in the treatment of conditions including but not limited to bone-related disease, bone formation, breast cancer (including those unresponsive to anti-estrogen therapy), cartilage formation, cartilage injury, cartilage loss, cartilage degeneration, cartilage injury, ankylosing spondylitis, chronic back injury, gout, osteoporosis, osteolytic bone metastasis, multiple myeloma, chondrosarcoma, chondrodysplasia, osteogenesis imperfecta, osteomalacia, Paget's disease, polymyalgia rheumatica, pseudogout, arthritis, rheumatoid arthritis, infectious arthritis, osteoarthritis, psoriatic arthritis, reactive arthritis, childhood arthritis, Reiter's syndrome, repetitive stress injury, periodontal disease, chronic inflammatory airway disease, chronic bronchitis, chronic obstructive pulmonary disease, metabolic syndrome, obesity, disorders of energy homeostasis, diabetes, lipid disorders, cardiovascular disorders, artherosclerosis, hyperglycemia, elevated blood glucose level, and insulin resistance.
X-ray crystal structures provide powerful tools for the rational design of ligands that can function as active agents for biologically important targets. The first crystal structure solved for ERR-α was a complex of the ERR-α ligand binding domain and a coactivator peptide from peroxisome proliferator-activated receptor coactivator-1 (PGC-1) (Kallen, Schlaeppi et al. 2004). The structure revealed that the putative ligand binding pocket (LBP) of ERR-α is almost completely occupied by side chains, in particular with the bulky side chain of Phe328. The crystal structure of ERR-α in a transcriptionally active conformation, in the absence of a ligand, provided evidence for ligand-independent transcriptional activation by ERR-α. A second ERR-α crystal structure was solved with the ligand binding domain of ERR-α (containing a C325S mutation) in complex with an inverse agonist bound in the ligand binding pocket (LBP). The C325S mutation was introduced to reduce biochemical instability problems during purification and crystallization that were determined to be associated with cysteine oxidation. (Kallen, Lattmann et al. 2007). The structure revealed a dramatic conformational change in the ERR-α LBP which created the necessary space for the ligand to bind. Due to the C325S mutation in the LBP, however, the structure left unresolved the importance of the Cys325 in designing ligands for use as modulators of ERR-α activity.
It has been shown that certain ligands form a covalent bond to a cysteine residue in the peroxisome proliferator-activated receptor (PPAR) ligand binding domain through a Michael addition, and that covalent binding is required for PPAR activation by the ligands (Shiraki, Kamiya et al. 2005). Covalent binding has also been demonstrated in a number of different drugs for a variety of drug targets. A few examples are briefly included below. It was proposed that targeted covalent inactivation of a variety of protein kinases may hold promise for developing treatments for a number of different diseases (US20060079494; Fry, Bridges et al. 1998; Schirmer, Kennedy et al. 2006; Wood, Shewchuk et al. 2008). Covalent binding was also demonstrated for potent and species-specific inhibitors of 3-hydroxy-3-methylglutaryl CoA synthases ((Pojer, Ferrer et al. 2006). It was shown that F-amidine and Cl-amidine irreversibly inactivate protein arginine deiminase 4 (PAD4) in a calcium-dependent manner via the specific modification of Cys645, an active site residue that is critical for catalysis. A growing body of evidence supports a role for PAD4 in the onset and progression of rheumatoid arthritis, a chronic autoimmune disorder. It was concluded that the covalent binding compounds may be useful as potential lead compounds for the treatment of rheumatoid arthritis (Luo, Arita et al. 2006). Even the unique properties of aspirin, the ubiquitous nonsteroidal anti-inflammatory drug, derive from its ability to covalently modify cyclooxygenases, COX-1 and COX-2, the in vivo targets for its action (Kalgutkar, Crews et al. 1998).
The present invention provides a crystallized form of a complex of the ERR-α ligand binding domain (ERR-α-LBD) with a ligand that forms a thioether bond to Cys325 of ERR-α. The diffraction pattern of the crystal is of sufficient resolution so that the three-dimensional structure of ERR-α can be determined at atomic resolution, ligand-binding sites on ERR-α can be identified, and the interactions of ligands with specific amino acid residues of ERR-α can be modeled and used to design ligands that can function as active agents. The assay methods of the present invention can be used to measure dissociation rates for ligands that form reversible covalent bonds and can function as active agents. Thus, the three-dimensional structure of the complex of the ERR-α ligand binding domain (ERR-α-LBD) with a ligand that forms a thioether bond to Cys325 and the assay methods of the present invention have applications to the design and biological characterization of ligands that function as modulators of ERR-α activity. Such ligands may be useful for treating, ameliorating, preventing or inhibiting the progression of disease states, disorders and conditions that are mediated by ERR-α activity.